The prevalence of autoimmune, immunologic and transplant-related diseases continues to increase while the knowledge of their pathogenesis and development of novel disease-modifying therapies lag behind. Over 5% of people in U.S. suffer from autoimmune diseases and up to 50% who received allogeneic hematopoietic cell transplantation (allo-HCT) will develop chronic graft-versus-host disease (cGVHD). Medical treatments remain unsatisfactory. Therefore, novel therapeutic and preventative approaches are desperately needed. The use of cell-based therapies has shown great promises for achieving the goals of prevention and cure. FOXP3+ regulatory T cells (Tregs) are central to the control of autoimmunity and maintenance of tolerance. Preclinical studies have convincingly demonstrated their therapeutic application for the prevention and treatment of multiple immune-mediated conditions. The critical next step is to translate those results into human treatments. A major hindrance is the lack of an efficient method to expand, isolate and characterize a highly purified Treg population of sufficient number for adoptive immunotherapy. To further develop on our established method for isolation of large quantity of highly purified Tregs from expansion cultures based on their selective expression of latency associated peptide (LAP), this project will address 1) the association and biomarkers of Treg repertoire, subsets and function for patients with allo-HCT who will develop cGHVD, 2) the function of CD121a and CD121b on LAP+ Tregs, and 3) the stability and potency of these LAP+ Tregs. The ultimate goal is to develop a LAP+ Treg product for clinical treatment of GVHD and other autoimmune conditions. Three specific aims have been established to accomplish these objectives. The first aim is to prospectively assess whether Treg repertoire, subset and function can predict patients with allo-HCT who will develop cGVHD. The outcome of this aim with provide justification for immunotherapy with Tregs to prevent or treat GVHD. The second aim is to decipher the role of CD121a and CD121b in maintaining Treg stability and suppressor function. The third aim will address the efficacy and stability of expanded LAP+ Tregs in vivo using a preclinical humanized murine model of GVHD. The successful accomplishment of these aims with provide the critical bridge for a clinical trial with LAP+ Tregs to treat or prevent cGVHD. The outcome of this project will make a significant advancement toward Treg adoptive immunotherapy and a major impact on clinical research for novel treatments of autoimmune, immunologic and transplant-related conditions by developing an efficient and reproducible method to consistently obtain highly purified Tregs and by providing quality assessment of the Treg products.